Metallocorrinoids are corrin rings with a metal-atom center, such as Co, Fe, Ni, or Mn. A corrin ring is four reduced pyrrole rings linked together A subclass of naturally occurring metallocorrinoids is known as cobalamin, that is, a cobalt-centered corrin ring. Naturally occurring vitamin B12, for example, is a cobalamin.
Vitamin B12 compounds are known to have many biological functions. They are required by the enzyme methionine synthase, for example, which is involved in the production of DNA. Pregnant women need increased amounts of vitamin B12 which is involved in the production of red blood cells. It is also believed that vitamin B12 enhances the effects of other vitamins and nutrients in tissue repair. Lack of vitamin B12 leads to megaloblastic anemia (characterized by large and immature red blood cells) and neuropathy in man with insidious onset of symptoms. These symptoms include weakness, tiredness, breathlessness (dyspnea) on exertion, tingling and numbness (paresthesia), sore tongue (glossitis), loss of appetite and weight, loss of sense of taste and smell, impotence, psychiatric disturbances (such as irritability, memory impairment, mild depression, hallucinations) and severe anemia (which may lead to signs of cardiac dysfunction). Deficiency of vitamin B12 leads to defective DNA synthesis in cells; tissues most affected are those with the greatest rate of cell turnover, e.g. the haematopoietic system. In small children Cbl deficiency can result in developmental delay, hematological disorders, and neurological disorders. There may be irreversible damage to the nervous system with specific demyelination of the spinal cord.
Increased availability of vitamin B12, on the other hand, appears to have a very beneficial effect. Cbl analogs and cobalamin drug conjugates have been shown to inhibit the growth of leukemia cells by possibly deactivating methionine synthase, thus preventing DNA synthesis. The cobalamins that are analogous to vitamin B12 compounds would appear to be potential therapeutic agents. These include hydroxocobalamin, cyanocobalamin, nitrocobalamin, mehtylcobalamin, and 5xe2x80x2-deoxyadenocobalamin, as well as nitrosylcobalamin.
All forms of vitamin B12 (adenosyl-, cyano-, hydroxo-, or methylcobalamin) are bound by the transport proteins intrinsic factor and transcobalamin II, to be biologically active. Those transport proteins involved in the uptake of vitamin B12 are referred to herein as cobalamin binding proteins. Specifically, gastrointestinal absorption of vitamin B12 relies upon the intrinsic factor-vitamin B12 complex being bound by the intrinsic factor receptors in the terminal ileum. Likewise, intravascular transport and subsequent cellular uptake of vitamin B12 throughout the body is dependent upon transcobalamin II and the cell membrane transcobalamin II receptors, respectively. After the transcobalamin II-vitamin B12 complex has been internalized, the transport protein undergoes lysozymal degradation, which releases vitamin B12 into the cytoplasm.
Cellular utilization of Cbl is preceded by two important receptor-mediated endocytic events. First, the dietary Cbl bound to gastric intrinsic factor (IF), a 50-kDa glycoprotein, is transported across the absorptive enterocyte via an intrinsic factor-cobalamin receptor that is expressed exclusively in the apical or the luminal membranes. The plasma transport of cobalamin to tissues/cells appears to occur via transporter transcobalamin II (TC II), by receptor-mediated endocytosis via transcobalamin II-receptor (TC II-R). Intracellularly released Cbl is then converted to its biologically active forms, (e.g. methyl-Cbl and 5xe2x80x2-deoxyadenosyl-Cbl) which are utilized by the cytoplasmic enzyme methionine synthase (MS) and mitochondrial at enzyme methyl-malonyl-CoA mutase (MMCM), respectively. MS activity is required for folate metabolism and DNA synthesis and presents a promising target to block cell proliferation. TCII and serum Cbl levels are both increased in hepatocarcinomas and leukemias. TCII has been identified as an acute phase reactant in autoimmune disorders and infection. Several studies have shown that high levels of Cbl inhibited L1210, P388D1, CCRF-CEM, and NCTC929 cell proliferation. This is likely due to the activation of an autoimmune response.
Recent studies have shown that TC II-R is expressed as a non-covalent homodimer of molecular mass of 124 kDa in tissue plasma membranes of human, rat, and rabbit. A comprehensive review of transcobalamin II, the transcobalamin II receptor, and the uptake of vitamin B12 is provided in xe2x80x9cTranscobalamin II and Its Cell Surface Receptor Vitamins and Hormonesxe2x80x9d, Vitamins and Hormones, Vol. 59, pgs. 337-366 (2000) which is incorporated herein in its entirety by reference thereto. Plasma membrane expression of TC II-R appears important for the tissue/cellular uptake of Cbl since its functional inactivation in vivo by its circulatory antiserum results in intracellular deficiency of Cbl. This intracellular deficiency in Cbl results in the development of Cbl deficiency of the animal as a whole.
The utilization of vitamin B12 as a delivery vehicle is known art. The art describes an oral delivery system that delivers active substances (hormones, bio-active peptides or therapeutic agents) by binding these agents to cobalamin or an analog thereof.
U.S. Pat. No. 5,936,082, which is hereby incorporated by reference in its entirety, for example, describes the therapeutic effectiveness of vitamin B12 based compounds. Nitrosylcobalamin (NO-Cbl), in particular, was evaluated for its chemotherapeutic effect. In five human hematological and eight solid tumor cell lines, NO-Cbl exhibited an ID50 that was 5-100 fold lower in tumor cell lines compared to benign cells (fibroblasts and endothelial cells). When oxidized from NO-Cbl, the NO free radical functions in a number of capacities. NO is involved in vasodilation, and is known to contribute to increased oxidative stress, inhibition of cellular metabolism and induction of DNA damage leading to apoptosis and/or necrosis.
Radiolabelled vitamin B12 analogs have also been described in the art as useful in vivo imaging agents. For example, U.S. Pat. No. 6,096,290, which is hereby incorporated herein in its entirety by reference thereto, describes the use of radiolabelled vitamin B12 analogs as in vivo tumor imaging agents.
U.S. Pat. No. 6,183,723, which is also incorporated herein by reference in its entirety, describes certain other cobalamin-drug conjugates.
The multiple components of Cbl uptake, enzymes, co-factors, and transport systems present several points of attack for the therapeutic delivery of cobalamins. As is described herein, the interrelationship of TCII-R and cytokines make this an attractive target for the therapeutic delivery of biologically active metallocorrinoids. Cytokines, in particular interferon xcex2, are shown to enhance the uptake or activity of biologically active metallocorrinoids, including vitamin B12 analogs, homologs, and derivatives.
Vitamin B12 analogs can be synthesized in a number of ways. In addition to conjugation of the side chains of the corrin ring, conjugation to the Cbl moiety can also be made, as can conjugation to the ribose moiety, phosphate moiety, and to the benzimidazole moiety. The conjugating agent and the drug to be conjugated depend upon the type of Cbl group that is modified and the nature of the drug. One of skill in the art would understand how to adapt the conjugation method to the particular Cbl group and drug to be coupled.
Preferred methods of attaching the drug to the Cbl molecule include conjugation to Cbl via biotin. Biotin is conjugated to either the propionamide or the acetamide side chains of the corrin ring of the Cbl molecule. The initial biotin-Cbl complex can be prepared according to Pathre, et al (Pathre, P.M., et al., xe2x80x9cSynthesis of Cobalamin-Biotin conjugates that vary in the position in cobalamin coupling, Evaluation of cobalamin derivative binding to transcobalamin II,xe2x80x9d incorporated by reference). Vitamin B12 is commercially available in its most stable form as cyanocobalamin from Sigma Chemical (St. Louis, Mo.).
One may most easily obtain transcobalamin II in the following manner: transcobalamin II cDNA is available in the laboratories of Drs. Seetharam (Medical college of Wisconsin) and Rothenberg (VA-Hospital, New York) TC II cDNA can be expressed in a Baculovirus system to make a large amount of functionally active TC II protein (see Quadros, E. V., et al., Blood 81:1239-1245, 1993). One of skill in the art would be able to reproduce the TC II cDNA. The antibodies to TCII-R were obtained through the laboratory of Dr. Bellur Seetharam, Med. College of WI.
One way to make cobalamin drug conjugates is through genetic engineering. In this method, a DNA sequence encoding TC II and the peptide drug may be expressed as one chimeric molecule. For example, it is possible to generate a chimeric construct using the full-length TC II cDNA and the cDNA for a peptide drug (e.g. insulin). The chimeric construct can then be expressed to produce a fusion protein consisting of the TC II-peptide drug. Following synthesis, the chimeric protein should be tested for both TC II activity and drug activity. Cobalamin can then be allowed to bind to this chimeric protein and used for therapy.
The observation that a cytokine (i.e. an interferon such as interferon-xcex2) upregulates or enhances the activity of the TCII-R provides a basis for a number of embodiments of the present invention.
One embodiment of the present invention is a method for increasing cobalamin-binding protein activity in a subject in order to treat a condition favorably affected by an increase in said cobalamin-binding activity, said method comprising the step of administering to a subject in need of such treatment a cytokine in an amount effective to increase cobalamin-binding activity in the subject. This method may further include the step of administering a vitamin B12 analog (which may be a naturally occurring vitamin B12 analog), nitrosylcobalamin or other suitable vitamin B12 drug conjugate. In this embodiment, the cytokine may be administered prior, simultaneously, or consecutively with the vitamin B12 analog. The cytokine and/or vitamin B12 analog may be administered prophylactically or acutely. The increased cobalamin binding protein activity is preferably TCII-R activity. The cytokine is preferably an interferon such as interferon-xcex2.
Another embodiment of the present invention is a composition that is comprised of a metallocorrinoid and a cytokine. It is preferable that the metallocorrinoid be a vitamin B12 analog, homolog, derivative or simply vitamin B12. This is particularly useful when there is a deficiency in vitamin B12 or if the vitamin B12 analog includes a drug conjugated thereto. It is particularly preferable that the vitamin B12 analog be a nitrosylcobalamin, but it may also be others known in the art, (e.g. hydroxocobalamin, cyanocobalamin, and methylcobalamin and 5xe2x80x2 deoxyadenocobalamin or radiolabelled cobalamin derivatives). The composition in accordance with this embodiment of the invention may also include a pharmaceutical carrier. It is preferable that the cytokine be an interferon, and more particularly interferon-xcex2.
Another embodiment of the present invention is a therapeutic composition comprising a cobalamin or cobalamin drug conjugate and a cytokine such as interferon-xcex2. In this embodiment, the therapeutic composition may also further comprise a pharmaceutical carrier. This is a particular advantageous embodiment when the cobalamin drug conjugate is designed for a specific aim in mind. Nitrosylcobalamin is just one cobalamin drug conjugate, and other drug conjugates may be selected from the group consisting of hydroxocobalamin, cyanocobalamin, methylcobalamin, and 5xe2x80x2 deoxyadenocobalamin, radiolabelled cobalamin, or other cobalamin and drug conjugate. This embodiment is particular useful in the treatment of diseases where the delivery of a therapeutic agent via a cobalamin delivery mechanism would be beneficial.
Another embodiment of the present invention is a method of enhancing uptake or activity of a metallocorrinoid comprised of administering a cytokine. It is preferable that the metallocorrinoid be a vitamin B12 or a vitamin B12 analog, homolog, or derivative. In this method it is preferable that the cytokine is an interferon, and more preferably that the interferon be interferon-xcex2.
Another embodiment of the present invention is a method of enhancing cellular uptake of a metallocorrinoid comprising the step of contacting a cell with a cytokine, particularly where the step of contacting a cell with a cytokine occurs through induction of cytokine. In this embodiment, it is preferable that the metallocorrinoid is vitamin B12 or a vitamin B12 analog. As in other embodiments, the vitamin B12 analog may he any suitable vitamin B12 analog, homolog or derivatives such as a cobalamin drug conjugate. In this embodiment it is preferable that the cytokine is an interferon, particularly interferon-xcex2.
Another embodiment of the present invention is a method of treating a patient comprising the steps of inducing cytokine production; and administering a metallocorrinoid. The step of inducing cytokine production may include administering a cytokine, or administering an agent as is known in the art to stimulate cytokine expression or production. The metallocorrinoid of this embodiment may be vitamin B12 or a vitamin B12 analog, homolog or derivative such as a cobalamin drug conjugate. The cytokine is preferably an interferon, more preferably interferon-xcex2.
Yet another embodiment of the present invention is a method of enhancing bio-availability of a metallocorrinoid, comprising the step of administering interferon-xcex2 alone or in combination with a metallocorrinoid.
Yet another embodiment of the present invention is a method of treating a subject to increase TCII-R activity in a cell comprising the step of administering to a subject in need of such treatment a cytokine to increase TCII-R activity in an amount effective to increase TCII-R activity in said cell. In this embodiment, it is preferable that the subject be cobalamin deficient. Another application of this embodiment is wherein the amount is sufficient to increase TCII-R activity above normal baseline levels. Preferably, this method may also be useful when the subject has an abnormally low level of TCII-R activity. This method preferably includes the step of co-administering a substrate (or ligand) of TCII-R, wherein the substrate of TCII-R is a cobalamin based compound (e.g. cobalamin or a cobalamin drug conjugate). The cobalamin drug conjugate is preferably nitrosylcobalamin, but may be any suitable cobalamin drug conjugate such as those known in the art.
Yet another embodiment of the present invention is a method of treating cancer comprised of administering a cytokine (e.g. interferon xcex2) to enhance the uptake or increase the availability of cobalamin analogs, homologs, or derivatives. This can be done either alone or in combination with the cobalamin analog, homolog, or derivative.
Another embodiment of the present invention is a method of imaging tissue or cells through enhanced uptake of radiolabelled vitamin B12 analogs, homologs or derivatives via administration of a cytokine such as interferon xcex2.
Additional aspects and applications of the present invention will become apparent to the skilled artisan upon consideration of the detailed description of the invention, which follows.